Influence of plant growth regulators,carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' (<Emphasis Type="Italic">Prunus avium</Emphasis> × <Emphasis Type="Italic">P. pseudocerasus</Emphasis>)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg l^–1 of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg l^–1 kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg l^–1) of chelated iron than that commonly used in tissue culture media (36.7 mg l^–1) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg l^–1 of 6-benzylaminopurine and 0.1 mg l^–1 of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg l ^–1 thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the emb-ryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.     

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l⁻¹ sorbitol, 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine and 2 g l⁻¹ Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine, 1.5 g l⁻¹ activated charcoal and 2 g l⁻¹ Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l⁻ sucrose, 20 g l⁻¹ Phytagel, 1.5 g l⁻¹ activated charcoal, 115.6 μM gibberellic acid and 214.8 μM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.     

Somatic embryogenesis in cell cultures of Thapsia garganica

Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l^–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EtOAc ethyl acetate - FDA fluorescein diacetate - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - 2-iP 2-isopentenyladenine - NAA 1-Napthaleneacetic acid     

Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Asexual embryogenesis and plant regeneration in Quercus

Red oaks (Quercus rubra L.) were regenerated via direct and indirect asexual embryogenesis from immature zygotic embryo tissues. Late heart and early cotyledonary explants cultured in light on modified MS medium proved to be most embryogenic. Embryoids arose from explants cultured on various combinations of 2,4-D and BA. However, the highest percentages of normal polar embryoids were produced by explants cultured on growth-regulator-free media. Epicotyl dormancy of embryoids was overcome by desiccation (air drying and use of an osmoticum) and rehydration treatments. Asexual plantlet development paralleled developmental changes associated with seed germination. White oak (Quercus alba L.) embryoids were also regenerated, but failed to germinate.     

Production potential of eicosapentaenoic acid by the diatom Nitzschia laevis

To investigate the production potential of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, the growth characteristics and fatty acid composition of the cells were studied under photoautotrophic, mixotrophic and heterotrophic conditions of growth. The specific growth rate and maximum biomass concentration were respectively 0.466 d^–1 and 2.27 g l^–1 for mixotrophic culture, 0.344 d^–1 and 2.04 g l^–1 for heterotrophic culture, and 0.167 d^–1 and 0.5 g l^–1 for photoautotrophic culture, respectively. As for EPA production, the yield and productivity were respectively 52.32 mg l^–1 and 10.46 mg l^–1 d for mixotrophic culture, 35.08 mg l^–1 and 6.37 mg l^–1 d for heterotrophic culture, and 6.78 mg l^–1 and 3.39 mg l^–1 d for photoautotrophic culture, respectively. Results suggest that mixotrophic culture is the most suitable growth mode for the production of EPA by the diatom Nitzschia laevis. The results are useful for the development of a cost-effective fermentation process for EPA production by Nitzschia laevis.     

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l^–1 to a chemically defined medium containing 140 g glucose l^–1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l^–1 to 47.7 g l^–1. With 40 g NaCl l^–1 at a dilution rate of 0.065 h^–1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

The production of 2,3-butanediol by fermentation of high test molasses

Summary Klebsiella oxytoca fermented 199 g·l^–1 high test or invert molasses using batch fermentation with substrate shift to produce 95.2–98.6 g 2,3-butanediol·l^–1 and 2,4–4.3 g acetoin·l^–1 with a diol yield of 96–100% of the theoretical value and a diol productivity of 1.0–1.1 g·l^–1·h^–1. Fermentation was performed numerous times with molasses in repeated batch culture with cell recovery. Such repeated batch fermentation, in addition to a high product yield, also showed a very high product concentration. For example, 118 g 2,3-butanediol·l^–1 and 2.3 g acetoin·l^–1 were produced from 280 g·l^–1 of high test molasses. The diol productivity in this fermentation amounted to 2.4 g·l^–1·h^–1 and can undoubtedly be further increased by increasing the cell concentration. Because the Klebsiella cultures ferment 2,3-butanediol at an extremely high rate once the sugar has been consumed, the culture was inhibited completely by the addition of 15 g ethanol·l^–1 and switching off aeration. Offprint requests to: A. S. Afschar     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

辣椒游离小孢子细胞团培养的胚状体形成

从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养．30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23％。显微镜检表明,这些胚状体来自游离的小孢子细胞．经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少．对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明．获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。     

Morphohistological and flow cytometric analyses of somatic embryogenesis in <Emphasis Type="Italic">Trifolium nigrescens</Emphasis> Viv.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l⁻¹ N⁶-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets.     

High frequency production of haploid embryos in asparagus anther culture

Summary A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l ^–1 casein hydrolysate, 800 mg l^–1 glutamine, 2 mg l ^–1 NAA, 1 mg l ^–1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l ^–1 NAA, 0.1 mg l ^–1 kinetin and 0.65 mg l ^–1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l ^–1 GA₃. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962)     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate) with flexible 3-hydroxyhexanoate content in<Emphasis Type="Italic"> Aeromonas hydrophila</Emphasis> CGMCC 0911

Aeromonas hydrophila CGMCC 0911 isolated from lake water was found to be able to synthesize a polyhydroxyalkanoate (PHA) copolymer (PHBHHx) consisting of 3-hydroxybutyrate (HB) and 4–6 mol% 3-hydroxyhexanoate (HHx). The wild-type bacterium accumulated 49% PHBHHx containing 6 mol% HHx in terms of cell dry weight (CDW) when grown on lauric acid for 48 h. When A. hydrophila CGMCC 0911 expressed the Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli, the recombinant strain could accumulate 47% PHBHHx, while the HHx content reached 17.4 mol%. The presence of changing glucose concentration in the culture changed the HHx content both in wild type and recombinant A. hydrophila CGMCC 0911. When 5 g l^–1 glucose was added to a culture containing 5 g l^–1 lauric acid as co-substrate, 45% PHBHHx/CDW consisting of 8.8 mol% HHx was produced by wild-type A. hydrophila CGMCC 0911 compared with only 5% in the absence of glucose. When the recombinant A. hydrophila CGMCC 0911 was grown on a mixed substrate containing lauric acid and 8–10 g l^–1 glucose, the HHx content could be further increased to 35.6 mol%. When the glucose concentration exceeded 10 g l^–1, cell growth, PHA content and mole percentages of HHx in PHBHHx were significantly reduced.     

Accumulation of astaxanthin and lutein in<Emphasis Type="Italic"> Chlorella zofingiensis</Emphasis> (Chlorophyta)

When grown photoautotrophically, Chlorella zofingiensis strain CCAP 211/14 accumulates a significant amount of valuable carotenoids, namely astaxanthin and lutein, of increasing demand for use as feed additives in fish and poultry farming, as colorants in food, and in health care products. Under standard batch-culture conditions, this microalgal strain exhibits high values of both growth rate (about 0.04 h^–1) and standing cell population (over 10¹¹ cells l^–1, or 7 g dry weight l^–1). Lutein, in a free (unesterified) form, was the prevalent carotenoid during early stages of cultivation (over 0.3 pg cell^–1, equal to 4 mg g^–1 dry weight, or 20 mg l^–1 culture), whereas esterified astaxanthin accumulated progressively, to reach a maximum (over 0.1 pg cell^–1, equal to 1.5 mg g^–1 dry weight, or 15 mg l^–1 culture) in the late stationary phase. A differential response of lutein and astaxanthin accumulation was also recorded with regard to the action of some environmental and nutritional factors. C. zofingiensis CCAP 211/14 represents a unique model system for analyzing the differential regulation of the levels of primary (lutein) and secondary (astaxanthin) carotenoids. Relevant also from the biotechnological viewpoint, this photosynthetic organism, with outstanding attributes for fast photosynthetic growth and carotenoid accumulation, might prove most valuable for its application to the mass production of either or both lutein and astaxanthin.     

Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding

Glycerol at 10–20 g l^–1 increased clavulanic acid production by Streptomyces clavuligerus in shake-flask culture. The biosynthesis of clavulanic acid continued for longer by feeding glycerol and production increased to 250 mg l^–1 compared with 115 mg l^–1 without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased further to about 280 mg l^–1 but also remained stable up to 130 h.     

Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.